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Near-infrared fluorescent probe for detecting alkaline phosphatase and manufacturing method thereof

a fluorescent probe and alkaline phosphatase technology, applied in the field of near-infrared fluorescent probes, can solve the problems of unresolved detailed mechanisms of alp activity regulation during pathogenesis, diverse physiological and pathological functions of alps, etc., and achieve the effect of selectively detecting alp only quickly and accurately

Active Publication Date: 2019-04-23
KOREA RES INST OF BIOSCI & BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present disclosure provides a fluorescent probe that can detect ALP through noninvasive in-vivo imaging. The probe has high selectivity and can be used for monitoring biological phenomena in cells and tissues. The disclosure also provides a method for preparing the fluorescent probe and a bone scaffold using the probe. The technical effects of the patent text include improved accuracy and noninvasive imaging for detecting ALP.

Problems solved by technology

Despite the extensive studies on ALP, its diverse physiological and pathological functions have not been fully elucidated.
Moreover, detailed mechanisms of ALP activity regulation during pathogenesis still remain unanswered.
This is in part a consequence of the absence of an appropriate probe for real-time tracking of ALP activity in living systems.

Method used

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  • Near-infrared fluorescent probe for detecting alkaline phosphatase and manufacturing method thereof
  • Near-infrared fluorescent probe for detecting alkaline phosphatase and manufacturing method thereof
  • Near-infrared fluorescent probe for detecting alkaline phosphatase and manufacturing method thereof

Examples

Experimental program
Comparison scheme
Effect test

synthesis example 1

nd Represented by Chemical Formula 15

[0103]Generally known 4-(((tert-butyldimethylsilyl)oxy)methyl)phenol (compound represented by Chemical Formula 12) was prepared.

[0104]Then, dichloromethane (DCM, 100 mL) was mixed with a mixture of the prepared 4-(((tert-butyldimethylsilyl)oxy)methyl)phenol (3.84 g, 16.1 mmol) and trimethylamine (TEA, 11 mL / 80.5 mmol) and then diethyl chlorophosphate (5.56 g, 32.2 mmol) was added. Then, the reaction mixture was stirred overnight at room temperature under argon atmosphere. The reaction mixture was diluted with DCM (100 mL), extracted with water (2×100 mL) and then washed with brine (100 mL). Then, the organic layer was dried with Na2SO4 and concentrated under reduced pressure to obtain an oily crude product (compound represented by Chemical Formula 14). The crude product was used in the following process without further purification.

[0105]The crude product obtained above was stirred together with EtOH (100 mL). Then, after adding HCl (8 mL) at roo...

synthesis example 2

nd Represented by Chemical Formula 5

[0110]Dichloromethane (DCM, 57 mL) was mixed with a mixture containing the prepared compound represented by Chemical Formula 15 (1.2 g, 4.61 mmol) and carbon tetrabromide (2.29 g, 6.92 mmol) and then triphenylphosphine (1.81 g, 6.92 mmol) was added at 0° C. Then, the reaction mixture was stirred at room temperature for 7 hours.

[0111]After removing the solvent by evaporating, the reaction mixture was purified by silica column chromatography using ethyl acetate / n-hexane (volume ratio: 1 / 2 to 1 / 1) as an eluent.

[0112]The result of 1H NMR and 13C{1H} NMR spectroscopy and mass spectroscopy of the finally obtained compound represented by Chemical Formula 5 ((4-(bromomethyl)phenyl diethyl phosphate), 1.3 g, 87%) is as follows.

[0113]1H NMR (CDCl3, 600 MHz): δ 7.38 (d, 2H, J=9.0 Hz), 7.20 (d, 2H, J=8.4 Hz), 4.48 (s, 2H), 4.24 (m, 4H), 1.37 (m, 6H) ppm.

[0114]13C{1H} NMR (CDCl3, 120 MHz): δ 150.67, 150.63, 134.46, 130.51, 120.30, 120.27, 64.70, 64.65, 32.68, ...

synthesis example 3

nd Represented by Chemical Formula 4

[0116]Generally known 6-methoxy-2,3-dihydro-1H-xanthene-4-carbaldehyde was prepared.

[0117]After dissolving the 6-methoxy-2,3-dihydro-1H-xanthene-4-carbaldehyde (200 mg, 0.82 mmol) in DCM (8.3 mL), a 1 M BBr3 DCM solution (20 eq, 16.5 mL, 16.5 mmol) was added at 0° C. and the mixture was stirred at 25° C. for 16 hours. Then, the mixture was added to a NaHCO3 solution at 0° C. and the aqueous layer was extracted with DCM / MeOH (10:1 mixture, 2×50 mL). Then, the organic layer was washed with H2O and dried with Na2SO4. Then, after removing the solvent, the product was dried in vacuo to finally obtain a compound represented by Chemical Formula 4 (6-hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde) as yellow solid (184 mg, 97%).

[0118]The result of 1H NMR spectroscopy and mass spectroscopy of the finally obtained compound is as follows.

[0119]1H NMR (DMSO-d6, 600 MHz): δ 10.18 (s, 1H), 7.19 (d, 1H, J=9.0 Hz), 6.92 (s, 1H), 6.61 (sd, 1H, J=2.4 Hz), 6.59 (dd, ...

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Abstract

A near-infrared fluorescent probe for detecting ALP is represented by Chemical 1. The fluorescent probe capable of detecting ALP can selectively detect ALP only quickly and accurately. In addition, the fluorescent probe allows monitoring of a biological phenomenon occurring in cells and tissues through noninvasive in-vivo imaging and during the early osteogenic differentiation in real time.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims, under 35 U.S.C. § 119, the priority of Korean Patent Application No. 10-2016-0162235, filed on Nov. 30, 2016, and Korean Patent Application No. 10-2017-0044300, filed on Apr. 5, 2017, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND1. Technical Field[0002]The present disclosure relates to a near-infrared fluorescent probe for detecting alkaline phosphatase (ALP) and a method for preparing the same.2. Background Art[0003]Alkaline phosphatases (hereinafter referred to as ‘ALPs’), which catalyze the hydrolysis and transphosphorylation of a wide variety of monophosphate esters, consist of a group of isoenzymes that are widespread in mammalian tissues (M. Syakalima et al., Jpn. J. Vet. Res., 1998, 46: 3; J. E. Coleman, Annu. Rev. Biophys. Biomol. Struct., 1992, 21: 441). The human genome encodes four different ALP enzyme isoforms, namely,...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07F9/06C12N9/18G01N21/64C07D209/10C09K11/06C07D311/80
CPCG01N21/6486C07D209/10C07D311/80C09K11/06C12N9/18C12Y301/03001C09K2211/1007C09K2211/1029C09K2211/1088A61K49/0019C12Q1/42G01N33/52G01N33/533G01N33/573
Inventor LEE, CHANG-SOOPARK, CHUL SOONKWON, OH SEOKHA, TAI HWAN
Owner KOREA RES INST OF BIOSCI & BIOTECH
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